BACKGROUND The focus of our research program is to understand the structural basis of muscle contraction at the molecular level. The benefits of the research are not limited to understanding muscle contraction, but the contractile system of muscle can serve as a prototype for many other motile processes in living cells, e.g. cell division and nutrient transport. The basic processes of muscle contraction are well understood: it is a result of cyclic interactions between myosin and actin, driven by the energy of actomyosin ATP hydrolysis. Muscle shortening involves the relative sliding of two sets of filaments: the thick, myosin containing filaments and the thin, actin containing filaments. Force is generated by myosin heads (cross-bridges) interacting cyclically with specific sites along the actin filaments. Since the availability of the crystal structures of the contractile proteins, and with the advent of single molecule assays, the field has made great strides in understanding the underlying processes. However, the details of the mechanism of transduction of chemical to mechanical energy still remains largely unresolved. One of the obstacles is that most of the studies at the molecular level are based on isolated, in vitro systems, e.g. the atomic structure of the myosin head is obtained alone without interacting with actin and EM reconstruction is based on isolated filaments. The link between the information obtained from the in vitro systems and the actual processes occurring intact muscle is still largely missing. The aim of our efforts is to provide such a link. Muscle can shorten for long distances, which result from the interactions between myosin heads and actin. It has been expected for sometime that some changes in the protein structures must take place to generate the movement. The atomic structures of the myosin head (S1) have revealed ligand-dependent differences. However, crystal structures of isolated S1 and an actin monomer alone do not necessarily reveal the in vivo structural changes that lead to force generation. To gain full understanding of the contraction mechanism, it is critical to determine the in vivo structures in a fully functioning muscle cell. X-ray diffraction from permeabilized muscle cells, the technique used in the present study, is one of the few techniques that reveal the structures as they occur in muscle cells, albeit at relatively low resolution. A wealth of structural information has been obtained. During the past several fiscal years, we have been systematically studying the distributions and orientations of the myosin cross-bridges in living muscle cells during the ATP hydrolysis cycle. Major characteristics of several the intermediate states have been determined. Objectives Our strategy over the past few years has been to characterize the structures of the individual hydrolysis intermediate states, such that structural transitions involved in force generation could be understood. To achieve this, we have used ATP analogues and chemically modified the myosins to "trap" the muscle in one of the intermediate states. To shorten the exposure time as much as possible so as to maintain viability of the muscle tissue, intense synchrotron radiation sources such as those at the Brookhaven National Laboratory have been routinely used as the X-ray source. Results The effects of temperature and ligand on the conformations in the myosin filament was further probed. During the previous fiscal year (FY2001) the experimental work was basically completed, showing that temperature and ligand has a profound effect on the distribution (disordered vs. ordered) of myosin heads in the filament. In FY02, upon detailed analysis of the experimental data, it was concluded that a helically ordered filament directly reflects that the myosin heads are in the "closed" conformation as determined by crystallography. Helical order can be used as a signature of the closed conformation in relaxed muscle. Furthermore, analysis shows that there are multiple conformations of myosin that exist in equilibrium with one ligand bound at the active site. Low temperature favors one disordered conformation while high temperature favors the ordered (closed) conformation together with a second disordered conformation. Significance The significance of finding is that for the first time an atomic structure is directly correlated with a myosin state in a living muscle cell. In addition, multiple conformations of myosin with one ligand bound breaks away from the conventional thinking of "one ligand, one conformation". This finding on myosin may be generalized to other proteins.